The regulation of redox homeostasis is essential for maintaining normal cellular functions such as signaling, growth, survival and death. Anomalous behavior of redox homeostasis adversely affects the normal physiological functions and is in turn responsible for numerous pathological conditions. Normally, cells in the diseased state exhibit high levels of aerobic glycolysis (Warburg effect), which results in oxidative stress. For example, the oxidative stress in cancer cells result in the accumulation of high levels of reactive oxygen species (ROS).
ROS constitute an important class of chemically reactive species that are essential for normal cellular functions including cell proliferation and differentiation. The optimum levels of ROS are controlled by various cellular redox homeostasis mechanisms, and an abrupt increase in their concentration levels is directly linked to oxidative stress-related disorders. Abnormally high levels of ROS are generated in response to adverse environmental and physiological stresses, exposure to ultraviolet (UV) light, and ionizing and heat radiations. It is crucial to monitor the levels of intracellular ROS for maintaining effective cellular homeostasis. Notably, different levels of ROS are responsible for different biological responses. Cell maintains different levels of ROS by activating the ROS-scavenging systems such as superoxide dismutases, glutathione peroxidase, redox enzymes (peroxiredoxins, glutaredoxin and thioredoxin) and catalase.
Mis-regulation in any of these ROS-scavenging processes leads to generation of excessive amounts of ROS. Accumulation of high levels of ROS causes oxidative damage of cellular components such as proteins, lipids and nucleic acids, which is responsible for ageing and many pathological conditions including cancer and cardiovascular, inflammatory and neurodegenerative diseases. It is known that cellular aging, also called cellular senescence, is a permanent cell cycle arrest state that results in increased production of ROS species. This increased ROS production is critical in maintaining the viability of the senescent cell. Therefore, it is necessary to develop molecular tools that are highly sensitive and can be activated by high levels of ROS to distinguish aged or diseased and normal cells.
ROS mainly comprises free radicals such as hydroxyl radical (OH.) and superoxide (O2.−) and reactive molecular species such as H2O2. H2O2 is one of the most prominent and essential ROS in biological systems and its significantly higher levels are generated in aged and cancer cells than in normal cells. In fact, H2O2 is a small molecular metabolite and plays a vital role in the regulation of various physiological processes in living organisms. Most importantly, H2O2 serves as a messenger in normal cellular signal transduction and is also a known marker for oxidative damage in many disease-associated cells. In cells, H2O2 is generated through the receptor-mediated NADPH oxidase (Nox) activation, which affects the functioning of signaling proteins that control cell signaling, proliferation, senescence and death.
The biological significance of H2O2 in human physiology and pathology has generated great interest in understanding the mechanistic details of H2O2 generation, partition and its role in cellular function and signaling pathways. In comparison to other ROS, relatively higher stability and diffusion rates of H2O2 through the plasma membrane makes it an attractive candidate to study its signaling pathways in living cells. However, the role of H2O2 as an essential messenger, for cellular signal transduction and its chemical reactivity and chemical instability limits its spatiotemporal tracking in real-time, especially in living cells. Molecular imaging of H2O2 using fluorescence probes is a highly attractive tool for studying its generation, accumulation, and trafficking and its role in biological processes in a spatiotemporal manner in living cells.
In recent years, stimuli-responsive fluorescence probes are gaining momentum due to their flexibility in introducing diversity through chemical modification and liberation of biologically active probes at the site of target cellular organelles, in response to biological analytes of interest.
Moreover, targeting specific subcellular organelles (mitochondria) and biomolecules such as DNA and proteins using stimuli-responsive fluorescence probes is an emerging powerful imaging technique that presents enormous potential in biomedical applications related to diagnostics and therapeutics. Therefore, there is a need in the art to develop such stimuli responsive fluorescence probes in order to carry out imaging, biomedical research, diagnosis, treatment etc.
Accordingly, the present disclosure provides for DNA-binding fluorescence probes with a stimuli-responsive appendage, wherein in response to a specific stimulus (chemical or enzyme), the appendage functionality is cleaved to release an NIR-fluorescence ready probe, which upon binding the minor groove of DNA fluoresces strongly, thus, aiding the imaging and quantification of the stimulus.